DESCRIPTION AAV vectors are exceptionally efficient for gene delivery to the brain by direct intraparenchymal infusion, where they mediate continuous transgene expression perhaps for the lifetime of the experimental animal. These exceptional properties of existing AAV vectors are the basis for ongoing or planned clinical trials for neurological diseases where focal gene delivery is therapeutically effective. However development of gene therapy approaches for many other neurological diseases will require global gene delivery to the CNS. Here we will use in vivo selection of an AAV capsid library and molecular grafting to develop new CNS- targeted AAV vectors with such capability. In the first approach we will use in vivo selection of an AAV capsid library with a diversity of 5x109 clones generated by DNA shuffling of AAV1, 2, 5, 8, 9, and rh10 Cap genes, to identify new brain- and spinal cord-tropic AAV capsids after intravenous or ICV delivery in adult animals. The library will be infused via the tail vein or the cerebral lateral ventricles of adult mice and one month later we will isolate DNA from the brain and spinal cord for PCR amplification of tissue-resident AAV Cap genes. These will be cloned back into the original library plasmid backbone and used to produce more AAV virions for subsequent rounds of in vivo selection. We will sequence 10 AAV Cap genes per round of selection. Once we determine convergence of tissue resident AAV Cap genes for each target, we will prepare recombinant AAV vectors encoding firefly luciferase (Fluc) using the newly selected capsids and infuse them into adult animals to determine their biodistribution. We will use bioluminescence imaging to assess distribution and kinetics of gene expression followed by biochemical quantification of Fluc activity in different organs and histological assessment of transduced cell distribution in brain and spinal cord. In the second approach we will combine AAV vectors with different proteins and peptides previously shown to be highly efficient in ferrying liposomes, enzymes, and siRNAs across the BBB after intravascular infusion. For this we will use chimeric AAV capsids carrying a 14 amino acid biotin acceptor peptide that allows for incorporation of biotin into the AAV capsid during packaging. As brain-targeting molecules we will use Streptavidin fused to a single-chain antibody specific for the mouse Transferrin receptor (TfR), or peptides derived from human ApoB-100, or Rabies virus glycoprotein. The biodistribution of the new AAV-molecular conjugates after intravenous infusion will be assessed as above. The therapeutic efficacy of all new CNS-targeted AAV vectors will be tested in a mouse model of GM1-gangliosidosis, which is a lysosomal storage disease that severely affects the CNS. We expect this new generation of CNS-targeted AAV vectors to foster the development of highly effective gene therapy approaches to treat many childhood, adult, and geriatric neurological diseases currently beyond the reach of modern medicine